Applications of the binding interaction of proanthocyanidins with bacteria and bacterial components

ABSTRACT

A composition having: a proanthocyanidin; and a macromolecule, an assembly of macromolecules, a semi-solid, or a solid surface to which the proanthocyanidini is immobilized.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/824,794, filed on Sep. 07, 2006, incorporated hereinby reference. U.S. patent application Ser. No. ______ to Delehanty etal., entitled “Binding Interaction of Proanthocyanidins with Bacteriaand Bacterial Components, filed on the same date as the presentapplication, and designated as 98833-US1 is incorporated herein byreference.

FIELD OF THE INVENTION

The invention is generally related to proanthocyanidins.

DESCRIPTION OF RELATED ART

Polyphenolic compounds are widely distributed in higher plants andconstitute a part of the human diet. Interest in polyphenolic compoundshas been spurred by their potential health benefits arising from theirantioxidant activity (Croft, Ann. NY Acad. Sci., 854, 435 (1998); Bravo,Nutri. Rev., 56, 317 (1998). All referenced publications and patentdocuments are incorporated herein by reference). The antioxidantactivity of flavanoids has been studied in great detail (Rice-Evans etal., Free Rad. Biol. Med., 20, 933 (1996); Cos et al., Planta Med., 67,515 (2001); Cos et al., J. Nat. Prod., 61, 76 (1998); Cos et al., InStudies in Natural Products Chemistry, Atta-ur-Rahrman, Ed., ElsevierScience B. V., Amsterdam (2000)). Tannins are an important group ofpolyphenolic compounds that are classified into three main groups: 1)the hydrolysable, 2) the complex, and 3) the condensed tannins orproanthocyanidins (PACs). PACs are high molecular weight polymerscomposed chiefly of the monomeric flavan subunits (+)-catechin and(−)-epicatechin and their derivatives whose structures consist of threephenyl rings each bearing various hydroxyl substituents (FIG. 1). PACsclassified as “type-B” are characterized by single linked flavanyl unitswhile “type-A” PACs contain an additional ether linkage between flavanylsubunits. Typical plant sources of PACs include fruits, leaves, andbark. In addition to their antioxidant activity, PACs have been shown topossess a number of other beneficial health effects includinganti-cancer activity (Zhao et al., Carcinogenesis, 20, 1737 (1999);Bomser et al., Chem.-Biol. Interact., 127, 45 (2000)), anti-inflammatoryactivity (Yang et al., J. Nutr., 128, 2334 (1998); Sen et al., Mol.Cell. Biochem., 216, 1 (2001)), and cardioprotective properties (Reed,Crit. Rev. Food Sci., 42S, 301 (2002)). Recently, significant attentionhas been placed on the health effects of PACs from green tea (Dufresneet al., J. Nutr. Biochem., 12, 404 (2001)), grapes (wines, juices, andgrape seed extracts) (Bagchi et al., Mut. Res., 523, 87 (2000)), andcranberry juice (Foo et al., Phytochemistry, 66, 2281 (2000)).Specifically, PACs from the American cranberry (Vaccinium macrocarpon)are well documented in their ability to protect the urinary tractagainst the adherence of uropathogenic bacteria and drinking cranberryjuice is a recommended treatment for various urinary tract infectionsand prostatitis. It has been shown that cranberry PACs inhibit theadherence of P-fimbriated Escherichia coli to cellular surfaces bearingα-Gal (1→4) β-Gal receptor sequences similar to those on epithelialcells of the urinary tract (Foo). This effect is mediated largely viaA-type PAC-induced conformational changes within the fimbriae proteinswhich undermine their ability to interact with cell surface receptors onuroepithelial cells (Howell et al., Phytochemistry, 66, 2281 (2005)).More recently, it has been shown that cranberry juice effectivelyreduces the adhesive forces between P-fimbriated E. coli and a siliconnitride probe surface (Liu et al., Biotech. Bioeng., 93, 297 (2006)).

Current strategies for filtering and/or concentrating bacteria andbacterial components are most often aimed at removing the materials fromsolutions through such non-selective means as size exclusion andelectrostatic interaction. Examples of sized exclusion-based andelectrostatic-based filter devices are Costar Corp.'s cellulose acetatefilters (size-based) with pore sizes of 0.22 μm to remove particleslarger than the size cutoff and Argonide Corp.'s NANOCERAM®electropositive nanometer aluminum oxide fibers (surface charge-based)that nonspecifically bind materials bearing a net negative surfacecharge (e.g., bacteria and viruses). More specific filtration andconcentration regimes utilize specific recognition elements (antibodies,peptides, aptamers, etc.) to specifically bind to molecules contained onor within the bacterial materials. Each of these technologies has itsown inherent limitations. Size exclusion and charge-based filtersrequire expensive manufacturing facilities and are often not re-usableonce a certain binding capacity has been reached. Filters andconcentrators based on specific recognition elements require theisolation of molecules with suitable binding characteristics followed bytheir large-scale preparation and purification.

Current therapeutic regimes for the neutralization and/or removal ofbacteria and bacterial components from host organisms (e.g., humans anddomestic livestock) are based largely on the use of antibiotics. Sincetheir introduction in the 1940's, antibiotic drugs have proven effectivefor the treatment of many bacteria-related illnesses. However, theirfrequent misuse has given rise to antibiotic-resistant bacterial strainsthat have necessitated the development and implementation ofincreasingly more powerful drugs. Further, while antibiotics effectivelyinhibit bacterial replication, they are often ineffective atneutralizing harmful bacterial toxins. For example, lipopolysaccharide(LPS), the major component of the outer leaflet of the outer cellmembrane of Gram-negative bacteria, is a major cause of complicationsduring bacterial infection. LPS, commonly referred to as bacterial“endotoxin,” is responsible for stimulating the body's normalinflammatory response against infection. Left unchecked, however, LPShyperstimulation can result in a life-threatening hyperactivation of theinflammatory cascade known as systemic inflammatory response syndrome(sepsis).

LPS is a complex glycolipid that comprises the major portion of theouter leaflet of the outer membrane of Gram-negative bacteria (Reatz,Ann. Rev. Biochem., 59, 129-170 (1990)). It is composed of two maindomains: 1) a lipid A core that is responsible for stimulating theimmune system through its interaction with Toll-like receptor 4 (TLR-4)and 2) an elongated, branched polysaccharide tail. A potent immuneresponse ensues upon the recognition of LPS by mammalian cells,including the production and release of cytokines, activation ofcomplement, and various other effects that result in the killing andclearance of the pathogen. Uncontrolled hyperinflammatory host responsesto LPS may lead to such life-threatening complications as septic shock,multiorgan failure, and even death. Polymyxin B (PB) is a cycliccationic antibiotic decapeptide that has been demonstrated to be one ofthe most efficient compounds exhibiting LPS-binding and outermembrane-disorganizing capabilities (Danner et al., Antimicrob. AgentsChemoth., 33, 1428 (1989)). PB is believed to inhibit the biologicalactivity of LPS via high-affinity binding to the lipid A moiety (Mooreet al., Antimicrob. Agents Chemoth., 29, 496 (1986)).

In light of the costs and limitations associated with currenttechnologies for the filtration and therapeutic neutralization ofbacteria and bacterial components, inexpensive alternatives to achievethese same tasks would be advantageous.

SUMMARY OF THE INVENTION

The invention comprises a composition comprising: a proanthocyanidin;and a macromolecule, an assembly of macromolecules, a semi-solid, or asolid surface to which the proanthocyanidin is immobilized.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention will be readily obtainedby reference to the following Description of the Example Embodiments andthe accompanying drawings.

FIG. 1 shows further proanthocyanidin structure. PACs are composed ofsubunits such as catecllin and epicatecllin. B-Type PACs contain asingle intermolecular bond either between carbons 4 and 8 or betweencarbons 4 and 6 while A-type PACs contain two intermolecular bondsbetween carbons 4 and 8 and carbon 2 and the oxygen of carbon 7 (Foo etal., J Nat Prod., 63, 1225 (2000)).

FIG. 2 shows the impact of cranberryjuice on non-specific adhesion. Thebackground intensity is expressed as the ratio of the mean backgroundintensity to the mean fluorescence signal intensity. Spiking ofbacterial samples with cranberryjuice (▴) and dialyzed filteredcranberry juice (◯) produces similar improvement in background signals.

FIG. 3 shows a schematic representation of agarose-PB bead pull-downassay. PB immobilized onto agarose beads are incubated withfluorescein-labeled LPS. After centrifugation and washing, the amount offluorescence associated with the beads is proportional to the amount ofLPS bound to PB.

FIG. 4 shows the interaction of PACs with LPS. (A) The data shows thepercentage of LPS bound to immobilized polymyxin B after co-incubationof LPS with PACs from cranberry, tea, and grapes. The LPS-bindingactivity of PACs from all three sources was concentration-dependent. (B)The data in (A) are presented as percent inhibition. (C) For cranberryPACs, the majority of the LPS-binding activity was contained within thefraction composed of polymers retained by 6,000 MWCO dialysis membranes(average degree of polymerization of 21). Data in A-C are themean±standard deviation and are representative of triplicateexperiments. PAC concentrations are reported in tannic acid equivalents.

FIG. 4 shows that cranberry PACs slightly reduce membrane binding of LPSand significantly inhibit LPS endocytosis. HEK 293 cells stablyexpressing CD14 and TLR4/MD2 were incubated with 25 nM LPS and 0.5 μMcranberry PAC for 1.5 h. Cells were either fixed (A) or fixed andpermeability (B) and incubated with a goat anti-LPS antibody conjugatedto fluorescein to visualize LPS. Where indicated, LPS binding wasfunctionally blocked by co-incubation with lipid A or anti-TLR4 andanti-CD14 antibodies. (A) PACs slightly inhibit the binding of LPS tothe cell surface. (B) PACs significantly abrogate endocytosis of LPS.The arrows indicate regions of internalized LPS. Nuclei are stained withDAPI. Quantitative analysis of LPS membrane binding and LPS endocytosisare shown in (C) and (D), respectively. Symbols correspond to levels ofsignificance relative to control (determined by Student's t-test): (*)p<0.1, (♦) p<0.05, (§) p<0.01, (□) p<0.001.

FIG. 6 shows that cranberry PACs inhibit endocytosis of LPS but do notinhibit overall endocytosis. Cellular binding assays were performed asdescribed in FIG. 2 in the manuscript except an Alexa Fluor 647-labeledtransferrin was added to the culture medium to label the endosomalcompartment. After fixation and permeabilization of the cells, LPS wasdetected with a goat anti-LPS antibody conjugated to fluorescein. Thedata show that in the absence of PAC (panel A), the endocytosed LPScolocalizes largely with transferrin (indicated by arrows in the mergedimage). In the presence of PAC (panel B), however, LPS is largelypresent at the plasma membrane (indicating an inhibition of LPSendocytosis by PAC). The transferrin staining of the endosomalcompartment, however, is similar to that seen in the absence of PAC.Thus, PAC inhibits LPS endocytosis but not overall endocytosis.

FIG. 7 shows that cranberry PACs inhibit LPS interaction with CD14 andTLR4/MD2 but not with LBP. (A) PACs completely inhibit binding of E.coli LPS to immobilized TLR4/MD2 (solid triangles) and partially inhibitbinding of LPS-FITC to immobilized CD14 (open squares). No inhibition ofLPS:LBP interaction was noted (solid circles). (B) PACs inhibit both thedirect and CD14-mediated binding of LPS-FITC to TLR4/MD2. In thepresence of 25 nM CD14, the binding of LPS to immobilized TLR4/MD2 isenhanced approximately 4-fold (open triangles) relative to when solubleCD14 is absent (solid triangles). The inset shows both data sets plottedas percent of control. In both instances, the degrees of inhibition toimmobilized TLR4/MD2 are comparable. Data are the mean±standarddeviation of two representative experiments.

FIG. 8 shows the inhibition of NF-κB activation by and cytotoxicity ofPACs in LPS-responsive HEK 293 cells. (A) Cranberry PACs inhibitLPS-induced NF-κB activation in a dose-dependent manner. (B) Theinhibitory effect of PACs is not overcome by excess LPS.HEK-CD14-TLR4/MD2 cells were stimulated with LPS at the indicatedconcentrations in the presence of cranberry PAC at the followingconcentrations: 0 nM (solid circles), 0.5 nM (solid triangles), or 10 nM(open triangles). (C) PACs are not toxic to LPS-responsive cells overthe same concentration range at which they inhibit NF-κB activation andPACs are ˜100-fold less toxic than LPS.

FIG. 9 shows the capture of FITC-LPS by immobilized proanthocyanidins.Sepharose-immobilized PACs from cranberries (open squares) and tea(solid circles) bind LPS in solution as indicated by the increase influorescence intensity in the pull down assay. The capture moleculeconcentration is 5.5 μM for PACs from tea and 6.0 μM for PACs fromcranberries

FIG. 10 shows the impact of soluble PAC presence on the immobilized PACcapture of LPS from solution. A. The presence of PACs in solutioninhibits the binding of LPS to Sepharose beads as indicated by thedecrease in fluorescence intensity upon increasing the PACconcentration. FITC-LPS concentration was 71 μg/mL and capture moleculeconcentration was 5.5 μM for PACs from tea (solid circles) and 6.0μM forPACs from cranberries (open squares).

FIG. 11 shows the impact of soluble lipid A presence on the capture ofLPS by immobilized PACs. Addition of Lipid A to PAC beads prior tocompletion of pull-down assays for the presence of FITC-LPS results in adecrease in the fluorescence intensity obtained. FITC-LPS concentrationwas 71 μg/mL and capture molecule concentration was 5.5 μM for PACs fromtea (solid circles) and 6.0 μM for PACs from cranberries (open squares).

FIG. 12 presents a comparison of proanthocyanidin capture of LPS to thatof polymyxin B. Immobilized PMB (solid triangles) and PACs from tea(solid circles) and cranberries (open squares) show similar bindingaffinities for FITC-LPS when compared in side-by-side assays. FITC-LPSconcentration was 145 μg/mL.

DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS

In the following description, for purposes of explanation and notlimitation, specific details are set forth in order to provide athorough understanding of the present invention. However, it will beapparent to one skilled in the art that the present invention may bepracticed in other embodiments that depart from these specific details.In other instances, detailed descriptions of well-known methods anddevices are omitted so as to not obscure the description of the presentinvention with unnecessary detail.

Grape seeds (Vitis vinifera) and white pine (Pinus maritima) are sourcesof proanthocyanidins, but the compounds are also found in food itemssuch as teas, coffees, chocolate, apples, berries, and barley, to name afew. PACs are found in heterogeneous mixtures consisting of variousnumbers of monomer subunits. Catechin and epicatechin are the mostcommon of the subunits. Intersubunit linkages are usually via singleintermolecular bonds between carbon atoms, but in some species subunitsare linked by two intermolecular bonds: one carbon-carbon and onecarbon-oxygen (FIG. 2) (Yoshida et al., J. Syn. Org. Chem. Jpn., 62, 500(2004); Foo et al., Phytochemistry, 54, 173 (2000)). These are referredto as B-type and A-type proanthocyanidins, respectively. Differingbiological activities have been shown for A-type and B-typeproanthocyanidins as well as for proanthocyanidins of differing subunitcomposition and differing degrees of polymerization (Kolodziej et al.,Phytother. Res., 9, 410 (1995); Howell et al., Phytochemistry, 66, 2281(2005)).

PACs from cranberries, tea, and grapes may bind efficiently to LPS fromvarious bacterial species. It is demonstrated that for cranberries, themost potent LPS-binding activity is contained within a PAC fractioncomposed of a mixture of polymers with an average degree ofpolymerization of twenty-one. While this fraction modestly inhibits thebinding of LPS to the surface of HEK 293 cells expressing the fullcomplement of LPS receptors (TLR4 (Toll-like receptor 4)/MD2 and CD14),it significantly abrogates the endocytosis of LPS. This PAC fractionalso inhibits LPS-induced nuclear factor-κB activation in a manner thatis not overcome by excess LPS. This effect is mediated through theinhibition of LPS interaction with TLR4/MD2 and the partial abrogationof LPS interaction with CD14. Importantly, PAC concentrations thatmediate effective LPS neutralization elicit minimal in vitrocytotoxicity. The results demonstrate the potent LPS binding andneutralization properties of PACs and identify PACs as a new class ofLPS antagonist with potential utility in endotoxin removal and the invivo treatment of sepsis.

LPS present in blood binds to LPS-binding protein (Tobias et al., J.Biol. Chem. 264, 10867-10871 (1989)), which transfers LPS to themembrane-anchored receptor, CD14, on mononuclear microphages. CD14 thenmediates the interaction of LPS with the bipartite receptor complex,Toll-like receptor 4/MD2 (TLR4/MD2), resulting in intracellularsignaling and production of nuclear factor-κB (NF-κB)-activatedinflammatory cytokines (Shimazu et al., J. Exp. Med., 189, 1777-1782(1999)). These cytokines include tumor necrosis factor alpha (TNFα) andinterleukins (IL-1α, IL-1β, and IL-6). Therapeutic strategies forsepsis, therefore, have been aimed at the neutralization of cytokines(Abraham et al., J.A.M.A. 273, 934-941 (1995)) or their receptors(Fisher et al., J.A.M.A. 271, 1836-1843 (1994)). Additional approacheshave focused on the neutralization of LPS with cationic compounds (Andraet al., J. Endotoxin Res. 12, 261-277 (2006)) or lipid A-like substances(Lien et al., J. Biol. Chem. 276, 1873-1880 (2001); Visintin et al., J.Immunol., 175, 6465-6472 (2005)). Unfortunately, clinical results forthese strategic avenues remain disappointing.

Proanthocyanidins (PACs) are plant-derived polyphenolic compoundscomposed of flavanoid subunits and they have recently been associatedwith several potential positive health benefits. Detailed studies haveattributed this activity to PACs with a degree of polymerization of 4 to5 containing at least one unique interflavan subunit linkage consistingof one carbon-carbon and one carbon-oxygen bond (referred to as anA-type linkage) (Foo). More recently, it has been shown that PACs induceconformational changes in bacterial P-fimbriae proteins that reducetheir adhesive forces for epithelial cell surface receptors (Liu).Recent work pointed to further interactions between high molecularweight polymers (average degree of polymerization of twenty-one) fromcranberry juice which inhibited the nonspecific adhesion of bacteria toa protein-functionalized immunosensor surface (Johnson-White et al.,Anal. Chem., 78, 853-857 (2006)). Based on this evidence, the potentialfor previously undescribed interactions of cranberry juice componentswith the bacterial cell surface were investigated.

The LPS binding properties of PACs from cranberries, tea, and grapes arereported herein. Focusing more closely on PACs from cranberries, theirability to bind LPS from multiple bacterial species, primarily throughinteraction with the conserved lipid A moiety, is demonstrated. ThePACs' ability to inhibit the interaction of LPS with cells expressingthe full complement of LPS receptors was determined. PACs inhibit LPSinteraction with mammalian cells largely through abrogation of LPSinteraction with TLR4/MD2, an activity that also mediates the inhibitionof LPS-induced NF-κB activation.

The interaction of PACs with bacterial cells for the prevention ofbacterial cell adhesion to proteins is demonstrated on glass surfacesused in immunoassay techniques. Further demonstrated is a heretoforeundescribed interaction of cranberry PACs with the bacterial cellsurface component, lipopolysaccharide (LPS; also known as bacterialendotoxin). The interaction of cranberry PACs with the P-type fimbriaeof E. coli has been described previously for the prevention of adhesionof bacterial cells expressing P-fimbriae to the cells of mammalianurinary and digestive tracts. The interaction of PACs withlipopolysaccharide has not been described previously. Based on thisactivity, PACs have potential uses in the filtration, concentration, andneutralization of bacterial endotoxins, bacterial cells, and bacterialcell components. Additionally, PACs may have uses in therapeuticapplications where the neutralization and/or removal of bacteria andbacterial cell components are warranted.

PACs may offer a number of advantageous new features that make themsuitable replacements or alternatives for current filtration,concentration, and neutralization applications. PACs are naturalproducts that are produced by higher plants. Hence, a theoretically“unlimited” supply is afforded by nature. They do not require expensivemanufacturing or machining facilities to produce as is the case withmanufactured filtration and concentration devices. All that is requiredis the extraction and purification of the natural materials in order toharness their benefit.

For therapeutic applications, PACs may be an attractive alternative tothe widespread use of antibiotics and current toxin-neutralizingcompounds. For example, antibiotics are the most common treatment forurinary tract infections and antibiotic spending to treat theseinfections currently totals more than $1.6 billion annually. Given themounting concern over the increase in antibiotic-resistant bacterialstrains, alternative therapeutic measures that alleviate the dependenceon antibiotics are being sought. The in vivo neutralization of bacterialtoxins with compounds such as polymyxin B and its derivatives iscurrently the recommended course of treatment to mitigate the onset ofimmune hyperstimulation during bacterial infections. However, theinherent toxicity of such compounds has limited their use and hasnecessitated the development of less toxic derivatives that retainefficacy. The PAC materials described herein, with relative affinitiescomparable to those of polymyxin B, may offer suitable alternatives fortoxin neutralization.

Interactions of PACs with P-fimbrae on cell surfaces are disclosed forthe reduction of cellular adhesion. Other potential interactions of PACswith various cell surface components provide potential mechanisms forthe neutralization of bacterial cells. Proteins such as cell surfaceprotein antigen and glucosyltransferases are involved in colonization.Other proteins are involved in various signaling pathways and in quorumsensing. Sugars on a cell's surface are also involved in communicationand anchoring and may serve as receptors. They are often the routethrough which pathogens such as viruses locate and interact with cells.The specific interaction between E. coli and PACs from cranberries notobserved with PACs from other juices indicates specificity in thefunction of various PAC species and indicates the potential for a rangeof interactions with cellular surface components that have not yet beenfully explored.

The PACs may be used in a composition, device, or component that may beuseful in filtering, detecting, removing, or otherwise interacting withbacteria and/or LPS. In such a composition, the PAC is immobilized to amacromolecule, an assembly of macromolecules, a semi-solid, or a solidsurface. The immobilized PAC may be exposed to a sample suspected ofcontaining LPS, lipid A, or a bacterium that produces LPS or lipid A. Inthe case of macromolecules, the PAC may be immobilized to themacromolecule by covalent bonding or any other form of bonding orforces. The macromolecules or assembly of macromolecules may, forexample, assist in transporting the PAC to a destination in an organismor be part of a sensor. Suitable macromolecules include, but are notlimited to, macromolecules that comprise an amino acid, a peptide, aprotein, a nucleotide, a nucleic acid, a lipid, or a carbohydrate.Suitable assemblies of macromolecules include, but are not limited to,those that comprise a multi-protein complex, a virus, a dendrimer, ananoparticle, a nanocluster, a nanocrystal, a nanorod, a nanosphere, ora nanotube. Solid and semi-solid surfaces may be useful in, for example,sensor and filters. Semi-solids include materials such as hydrogels.Suitable solid or semi-solid surfaces include, but are not limited to, aplurality of beads, a sphere, a rod, fibers, filaments, capillaries, atube, a planar layer, or a waveguide.

The degree of polymerization of the PAC may be selected depending on theparticular application. Suitable average degrees of polymerization ofall the PACs in a composition include, but are not limited to, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. The PAC maycontain only catechin and epicatechin units or may contain otherflavanoid units.

The PACs may be used in a filter apparatus. Such an apparatus maycomprise a housing having an inlet and an outlet to allow fluid flowthrough the housing and the immobilized PAC. The immobilized PAC ismaintained within the housing when fluid is flowed through the housing.This may be useful for removing bacteria and/or LPS from a fluid such asblood. An infected patient's blood may be passed through the filter andinjected back into the patient. The filter may also be useful fordecontamination and purification.

The PACs may be used in a sensor apparatus. Such an apparatus maycomprise a fluid flow apparatus, the immobilized PAC, and a mechanismfor detecting the binding of LPS, lipid A, or bacteria to the PAC. Themechanism may be any mechanism used in sensors for detection of abinding event, including but not limited to, an optical mechanism,ultraviolet light absorbance, visible light absorbance, infraredabsorbance, fluorescence, luminescence, chemiluminescence, polarization,surface plasmon resonance, changes in refractive index, an acousticmechanism, a surface acoustic wave device, a quartz crystal microbalancedevice, an electrochemical mechanism, or amperometric, potentiometric,or conductimetric measurements.

A filtering or sensing apparatus may be used with a variety of samples,including but not limited to, a clinical sample, blood, plasma, serum,lymph, spinal fluid, a pharmaceutical preparation (which may requiredecontamination) or a food or beverage intended for infants orimmunosuppressed individuals.

Several example strategies for immobilization of PACs are describedherein.

(1) Direct reactivity of PAC hydroxyls—The hydroxyls within PACs reactsimilarly to amino groups toward acylating agents, compounds thatcontain an activated acyl group where the nucleophile attacks at thecarbon displacing a leaving group. Acylating agents include, but are notlimited to, the acid anhydrides, isocyanates, isothiocyanates,imidoesters, acid halides, N-hydroxysuccinimdyl and other activatedesters. Thus, crosslinking agents bearing one of these acylating groupson one end (directed at the PAC hydroxyls) and another functional group(directed toward reactive groups on the targeted surface ormacromolecule) are suitable for the immobilization of PACs. A number ofsuch crosslinkers are available commercially (see, for example,www.piercenet.com). Three examples of direct reactivity are describedbelow.

(1A) Immobilization of Proanthocyanidins on sulfhydryl-bearingmacromolecules. Purified PACs can be immobilized onto proteins or othersulfhydryl-bearing macromolecules and surfaces through the use ofN-[p-maleimidophenyl] isocyanate (PMPI, Pierce). Incubation (45 min atroom temperature) of PACs with PMPI at a molar ratio of 1:10 in 10 mMborate buffer at pH 8.5 results in reaction of the isocyanate group ofPMPI with the hydroxyl groups of the PACs to produce carbamate linkages.Addition of the sulfhydryl-bearing compound and sodium phosphate buffer(pH 7, final concentration 50 mM) results in reaction of the maleimidegroup of PMPI with the sulfhydryl groups of the proteins or othermacromolecules. The concentration of the macromolecule can be varied toinfluence the number of PACs bound to each molecule.

(1B) Immobilization of proanthocyanidins on amine-bearingmacromolecules. Purified PACs can be immobilized onto proteins or otheramine-bearing macromolecules and surfaces through the use of4-(chlorosulfonyl) phenyl isocyanate (CSPI). Incubation (45 min at roomtemperature) of PACs with CSPI at equimolar concentrations in boratebuffer at pH 8.5 results in reaction of the isocyanate groups of CSPIwith the hydroxyl groups of the PACs to produce carbamate linkages.Addition of the amine-bearing macromolecule then allows the reaction ofthe chlorosulfate group of CSPI with surface amine groups to proceed,producing sulfonamines through the mechanism employed by the HinsbergTest.

(1C) Immobilization of proanthocyanidins on amine or carboxyl-bearingmacromolecules. Amine or carboxyl-bearing macromolecules can be modifiedthrough the use of 3-[(2-aminoethyl)-dithio]propionic acid•HCl (AEDP) inthe presence of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC). The use of a reducing agent such astris[2-carboxyethyl]phosphine hydrochloride (TCEP) or2-mercaptoethylamine cleaves the S—S bond of the AEDP producing asulfhydryl group. A PMPI crosslinker can then be used to bindproanthocyanidins to this sulfhydryl group as described above (1A).

(2) Conversion of hydroxyls to alternative functional groups. (2A)Conversion of hydroxyls to aldehydes. The vicinal hydroxyl groups ofPACs are susceptible to oxidation with periodic acid, sodium orpotassium periodate. Periodate oxidation cleaves C—C bonds bearingadjacent hydroxyls, converting them to dialdehydes (Bobbit, Adv.Carbohyd. Chem., 11, 1 (1956)). After periodate treatment, thedialdehyde formed can react with a variety of reagents, notably withamine groups to form imines of Schiff bases.

(2B) Conversion of hydroxyls to sulfhydryls. PAC hydroxyls can beconverted into sulflydryl groups. The hydroxyl group can be activatedwith tosyl chloride (toluenesulfonyl chloride) in phosphate buffer (pH 8to 9) containing dioxane or pyridine. Subsequent trans-esterification isachieved in 0.5 M thioacetate solution at pH 5.5. Hydrolysis of thethioester to generate a free thiol is done with 0.5 N methanoate.

The properties described here show that PACs may be useful in treatingpatients diagnosed with a gram negative bacterial infection or sepsis orimmunosuppressed patients. Any pharmaceutically acceptable treatmentusing a composition comprising a PAC may be used. The treatment may bemore effective when performed in a combination therapy with anantibiotic, a chemotherapeutic, a radionucleide, an immunosuppressivedrug, a plasmapheresis treatment. The PAC may also be conjugated to anantibiotic, a chemotherapeutic, a radionucleide, or an immunosuppressivedrug.

One example application is binding of LPS, lipid A, or bacterial cellsfor their removal. The immobilization of PACs to any number ofmacromolecules, assembly of macromolecules, or surfaces would allow PACsto be used as recognition elements for the 1) removal, 2) concentration,and 3) purification of lipopolysaccharide, lipid A, or bacterial cellsfrom solutions where their presence is not desired. This binding cantake either of two forms: direct or indirect. The direct bindingembodiment applies to instances in which the PACs are immobilized tosolid and/or semi-solid surfaces (comprising beads, spheres, rods,fibers, filaments, capillaries, tubes, planar layers, or waveguides).Herein, the bead-immobilized PACs capture the target lipopolysaccharide,lipid A, or bacterial cells and the resulting complexes are centrifugedor magnetically concentrated such that the supernatant is easilyremoved, resulting in solid support-associated LPS, lipid A, orbacterial cells. Alternatively, PACs immobilized in this way can be usedin a column format, wherein a packed column of PAC-functionalizedsupport is exposed to a solution containing LPS, lipid A, or bacterialcells. Indirect embodiments involve the immobilization of PACs tomacromolecules and assemblies of macromolecules. These are used to bindthe LPS, lipid A, or bacterial cells in solution. After binding,addition of solid-support-immobilized recognition elements directedagainst the macromolecules or assemblies of macromolecules to which thePACs are attached allow for capture of the complexes and their removalfrom solution. In this scenario, the macromolecule used can be comprisedof any of the following: an amino acid, a peptide, a protein, anucleotide, a nucleic acid, a lipid, or a carbohydrate. The assembliesof macromolecules can be comprised of any of the following: amultiprotein complex, a virus, a dendrimer, or a nanoparticle. Thenanoparticles can comprise a nanocluster, a nanocrystal, a nanorod, ananosphere, or a nanotube.

Another example application is binding of LPS, lipid A, or bacterialcells for their detection. PACs' ability to bind LPS, lipid A, orbacterial cells can also be used to achieve the detection of LPS, lipidA, or bacterial cells once they are bound. Essentially, a sensor basedon immobilized PACs comprises the immobilized PACs (as described above),a fluid flow apparatus, and a mechanism for achieving the detection ofthe bound LPS, lipid A, or bacterial cells. The detection can comprisethe following methods: optical (UV or infrared absorbance, fluorescence,luminescence, chemiluminescence, polarization, surface plasmonresonance, changes in refractive index) acoustic (surface acoustic wave,quartz crystal microbalance), electrochemical (amperometric,potentiometric, conductimetric).

Another example application is binding of LPS, lipid A, or bacterialcells for therapeutic applications. The envisioned therapeuticapplications of PACs are both in vivo and ex vivo. The in vivoapplications comprise the administration of PACs (either passivelythrough the diet or actively through bolus pill form or injection) topatients who are at risk of or are suspected to have a bacterialinfection. The use of PACs in combination with other establishedantibacterial, antiviral, and antifungal methods is also envisioned.Such combination therapies comprise the simultaneous administration ofPACs along with traditional antibiotic, antifungal, or antiviralmedications. The direct immobilization (via the methods described above)of PACs to these types of medications

The ex vivo applications comprise the use of PACs in a number offormats. These include the topical application of PACs to areas of theskin that are susceptible to bacterial infection. These also include useof PACs in plasmapheresis applications, wherein a patient's plasma isremoved, incubated with PACs to remove LPS, lipid A, and bacterial cellsand then the plasma is subsequently restored to the patient.

The presence of LPS during bacterial infection is a primary cause ofsepsis, a severe inflammatory condition for which effective therapiesremain limited. Among the more recent strategies for the neutralizationof LPS are the use of natural and synthetic cationic compounds and lipidA-like substances. Cationic compounds, including peptides based on thebinding domains of natural LPS-binding proteins and antimicrobialpeptides, have exhibited affinities for LPS in the nanomolar range(Andra et al., J. Endotoxin Res., 12, 261-277 (2006)). The cyclicnonapeptide polymyxin B, which binds to the lipid A moiety of LPS, hasreceived considerable attention for its potent LPS neutralizing activity(David et al., Biochim. Biophys. Acta., 1165, 147-152 (1992)). However,its clinical utility has been hampered by its considerable toxicity.Lipid A-like substances, which mimic the conserved lipid A moiety,function by preventing the interaction of LPS with its receptors. Forexample, E5564 is a second-generation synthetic lipodisaccharidedesigned to abrogate LPS interaction with the TLR4/MD2 receptor (Lien etal., J. Biol. Chem., 276, 1873-1880 (2001); Visintin et al., J.Immunol., 175, 6465-6472 (2005)). Despite their potent LPS neutralizingactivity, cationic compounds and lipid A-like substances are oftenlimited by either their toxicity or their ability to be overcome by highLPS concentrations (Golenbock et al., J. Biol. Chem., 266, 19490-19498(1991)). LPS binding compounds that overcome these deficiencies areneeded.

Described herein is a previously unreported biological activity ofnaturally-occurring plant PACs: the efficient binding and neutralizationof bacterial LPS. The results demonstrate that PACs from cranberries,tea, and grapes bind LPS in a dose-dependent manner. Further, in thecase of cranberry PACS, larger polymers (with an average degree ofpolymerization of twenty-one) exhibit the most potent LPS-bindingactivity.

Previous work has established the important role of PAC interaction withcomponents of the bacterial cell surface. Foo et al. demonstrated thatLH20-purified PACs from cranberries inhibited the adherence ofP-fimbriated E. coli to surfaces containing α-Gal(1→4) β-Gal receptorsequences (Foo et al., Phytochemistry, 54, 173-181 (2000)). Thisactivity was associated with PACs with a degree of polymerization of 4to 5 and bearing at least one A-type linkage. Howell et al. laterreported that this effect was, indeed, specific to A-type linkages, asB-type linked PACs from various sources did not mediate the effect(Howell et al., Phytochemisty, 66, 2281-2291 (2005)). More recently, Liuand coworkers proposed a mechanism for the PAC-mediated decrease ofbacterial adhesion. Atomic force microscopy (AFM) studies showed thatPACs induced a shortening of the P-fimbriae proteins, resulting inreduced adhesive forces between the bacterium and the AFM probe tip (Liuet al., Biotechnol. Bioeng., 93, 297-305 (2006)).

In contrast to the nature of PACs' interaction with P-fimbriae proteins,the present findings point to several differences with respect to thenature of PACs' recognition of LPS. First, LPS binding is not specificto the A-type interflavan linkage. PACs from cranberries contain both A-and B-type interflavan subunit bonds while tea and grape PACs containexclusively B-type interflavan linkages. Still, PACs from all threesources efficiently bound LPS. Second, data obtained for cranberry PACsdemonstrated that larger polymers (fraction with a degree ofpolymerization of twenty-one) possess the highest degree of LPS bindingactivity. Finally, while other studies on the anti-bacterial adhesionproperties of PACs have concentrated almost exclusively on PACs' effectson uropathogenic E. coli, the present data clearly shows that cranberryPACs bind LPS from multiple Gram-negative bacterial species. Further,binding studies showed that cranberry PACs recognize mutant LPS bearingshorter polysaccharide chains as well as diphosphoryl lipid A with anaffinity comparable to that for native LPS. Thus, the lipid A moietyplays a predominant role in PACs' recognition of LPS.

The LPS binding activity of cranberry PACs' can have a significantimpact on the interaction of LPS with LPS-responsive cells. In thecurrent model of cellular interaction with LPS, LPS binding protein(LBP) present in serum binds to and presents LPS to themembrane-resident receptor CD14, which in turn transfers LPS to thebipartite receptor complex, TLR4/MD2 (Shimazu et al., J. Exp. Med. 189,1777-1782 (1999)). MD2 is the LPS-binding unit of the receptor whileTLR4 serves as the signal transduction component (Shimazu; Nagai et al.,Nat. Immunol., 3, 667-672 (2002); Schromm et al., J. Exp. Med., 194,79-88 (2001)). The TLR4/MD2-LPS complex ultimately undergoes endocytosisinvolving a caveolae-dependent uptake mechanism as part of LPS-inducedreceptor down-regulation (Shuto et al., Biochem. Biophys. Res. Commun.,338, 1402-1409 (2005)). While debate currently exists as to whether TLR4physically contacts LPS, it is clear that TLR4/MD2 and LPS form a stablecomplex on the cell surface and that LPS binding to MD2 is aprerequisite for TLR4 signaling activity (Visintin et al., J. Immunol.,175, 6465-6472 (2005)) and LPS endocytosis (Husebye et al., Embo. J, 25,683-692 (2006); Shuto et al., Biochem. Biophys. Res. Commun., 338,1402-1409 (2005)).

In HEK 293 cells expressing the full complement of LPS receptors, it wasfound that cranberry PACs modestly reduced the binding of LPS to thecell surface while they significantly inhibited LPS endocytosis. Inmembrane binding experiments, cranberry PACs were slightly more potentat abrogating LPS interaction with the cell membrane than wasdiphosphoryl lipid A, a known MD2 receptor agonist and LPS antagonist(Muroi et al., J. Biol. Chem., 281, 5484-5491 (2006)). PACs were lessefficient than a CD14 function perturbing antibody at abrogating LPSbinding. This is consistent with CD14's established role as the initialpoint of membrane interaction for LPS. An inhibitory effect of cranberryPACs on the endocytosis of LPS nearly equivalent to that of lipid A wasnoted in internalization assays. Based on these results and the knowndependence of LPS endocytosis on efficient LPS interaction with MD2(Shuto et al., Biochem. Biophys. Res. Commun., 338, 1402-1409 (2005)),it can be reasoned that cranberry PACs played a predominant role inabrogating LPS interaction with the TLR4/MD2 receptor complex. Indeed,receptor binding studies showed that while PACs achieved a maximal 40%inhibition of LPS interaction with CD14, they completely inhibited LPSbinding to TLR4/MD2 over the same concentration range. Further, PACsinhibited NF-κB activation in a manner that was not overcome by high LPSconcentrations, even when present at a six-fold molar excess over PACs.

Other reports have described the inhibition of LPS-induced production ofinflammatory cytokines by PACs. Bodet et al. demonstrated that aPAC-enriched fraction from cranberry juice concentrate inhibited theLPS-induced production of IL-6, IL-8, and prostaglandin E2 in gingivalfibroblasts (Bodet et al., Eur. J. Oral. Sci., 115, 64-70 (2007)) andTNFα and RANTES (Regulated on Activation Normal T-cell Expressed andSecreted) in macrophages (Bodet et al., J. Dent. Res., 85, 235-239(2006)). While this fraction was shown to inhibit the phosphorylationstate of intracellular signaling proteins, the exact mechanism of thesignaling inhibition was not elucidated. The present findings, however,point to a mechanism of LPS inhibition in which PACs bind directly toand neutralize LPS by blocking its interaction with TLR4/MD2. Further,in contrast to other LPS binding substances, this interaction with LPSis not readily overcome by excess concentrations of LPS. This hasremained a shortcoming in the development of other LPS bindingsubstances as therapeutic reagents for sepsis. The results suggest,therefore, that the multivalent nature of the PAC polymeric structureallows for other stabilizing interactions with the polysaccharide chainof LPS.

PACs are structurally quite different from other previously describedLPS scavengers or LPS receptor antagonists such as polymyxin B or lipidA-like substances. Further, their LPS binding properties suggest thatthey may hold promise for the development of new therapies for the invivo treatment of sepsis. At a minimum, their potent LPS bindingactivity, low cost, and widespread availability from natural sourcesshould make them useful in biotechnology applications wherein theremoval and purification of endotoxin is required.

Having described the invention, the following examples are given toillustrate specific applications of the invention. These specificexamples are not intended to limit the scope of the invention describedin this application.

EXAMPLE 1

Purification of Proanthocyanidins—Dialyzed cranberry juice concentrate(DCC) was produced from Mountain Sun pure unsweetened cranberry juice(100% strength, Celestial Group, Inc.) by dialysis against water (6,000MWCO dialysis tubing) and filtration through a 0.2 μm filter. PACs (inwhich nonspecific polyphenols have been removed) were obtained fromwhole cranberry juice, Welch's 100% red grape juice, or Lipton black teavia purification by hydrophobic adsorption chromatography using aSephadex LH20 column (Hagerman, “The tannin chemistry handbook”http://www.users.muohio.edu/hagermae/tannin.pdf (2002)). Whole juice wasreduced by rotary evaporation to a minimum volume and resuspended to theoriginal volume in 70% acetone, sonicated for 30 min, and filtered withWhatman #3 filter paper. Resuspension, sonication, and filtration of theinsoluble material was repeated twice more and all liquid was combined.This solution was reduced by rotary evaporation to remove all acetoneand resolubilized in 75% ethanol to twice the original volume. Tea wasextracted by sonication of one family sized tea bag in 200 mL 70%acetone for 20 mim (repeated 3 times). For each preparation, thesolutions were combined, reduced by rotary evaporation, andresolubilized in 200 mL 75% ethanol. This solution was applied to aSephadex LH20 column in batches equal to the bed volume. Small phenolicswere removed by elution with ethanol equivalent to five times the bedvolume. PACs were eluted with acetone and reduced by rotary evaporationto a minimum volume. PACs recovered from whole cranberry juice weresubsequently fractionated by differential dialysis against watercontaining 25% ethanol for further characterization. Fractions werecollected as those which pass through 2,000 MWCO tubing (Spectra/Por;Dial<2k); those which pass through 3,500 MWCO tubing (Spectra/Por CE)but are retained by the 2,000 MWCO (Dial 2-3k); those that pass through6,000 MWCO tubing (Spectra/Por Membrane MWCO 6-8000) but are retained bythe 3,500 MWCO tubing (Dial 3-6k); and those which are retained by the6,000 MWCO tubing (Dial 6k). All materials were dried to powder under anitrogen stream for storage. Purified materials were dissolved in 33%ethanol/H₂O for use in binding experiments. The degree of polymerizationof each purified compound was determined by modified vanillin assaycombined with the acid butanol assay PAC concentrations were determinedby radial diffusion assay using tannic acid as a standard (Hagerman,“The tannin chemistry handbook”http://www.users.muohio.edu/hagermae/tannin.pdf (2002); Hagerman, J ChemEcol. 1987, 13, 437) Analysis of purified materials by thiolysis andHPLC indicated no low molecular weight species remaining following LH20separation (Hammerstone et al. J. Nutr. (2000) 130: 2086S-2092S; Gu etal., J. Agric. Food Chem. (2002) 50: 4852-4860; Prieur et al.,Phytochemistry (1994) 36:781-784; Sun et al., J. Agric. Food Chem.(1998) 46:1390-1396). On the basis of these analyses, the PACs wereconsidered to be devoid of sugars, acids, and low molecular weightcontaminants.

TABLE 1 Proanthocyanidin Specifications Tannic Acid Equiv. (μM) AverageDegree of PAC Source (1 mg/mL) Polymerization Cranberries 63.4 12.6Black Tea 49.8 4.1 Grape Juice 29.9 7.2 Cranberry Juice 39.0 8.9Cranberries, Fraction 1 38.3 21.7

EXAMPLE 2

Non-specific Adhesion of E. coli—The Naval Research Laboratory ArrayBiosensor employs a protein-coated glass waveguide for the detection ofanalytes of interest (Rowe et al., Anal. Chem., 71, 433 (1999); Taitt etal., Microbial. Ecol., 47, 175 (2004); Golden et al., Talanta, 65, 1078(2005)). The surface of the waveguide has a patterned array of capturemolecules with non-specific passivating molecules used to coat otherregions of the surface (Sapsford et al., Anal. Chem., 74, 1061 (2002);Ngundi et al., Anal. Chem., 77, 148 (2005); Shriver-Lake et al., Anal.Chem., 67, 2431 (1995)). Fluorescence-based detection of targets isdependent on discrimination of capture molecule areas from other areasof the waveguide. Non-specific adhesion of targets to unexpected areasof the surface negatively impacts limits of detection as well as falsepositive/negative rates for the Array Biosensor.

The combination of a nonpathogenic E. coil strain (ATCC 35218) and a lowaffinity antibody (rabbit polycolonal antibody to E. coli; Abeam, Inc;Cambridge, Mass.) was found to produce a degree of nonspecific bindingwhich made discrimination of signal from background intensitiesdifficult (Johnson-White et al., Anal. Chem., 78, 853 (2006)). Bacterialcells adhere through interactions involving surface proteins and/orlipopolysaccharide (LPS). Traditional approaches to reduction ofnonspecific binding (for example blocking waveguide surfaces or spikingsamples with proteins or sugars) were unsuccessful. Based on the impactof cranberryjuice on bacterial cell adhesion in the urinary tract, thejuice was investigated as a potential mediator of adhesion in the ArrayBiosensor. FIG. 2 presents data on the ratio of background intensity tosignal intensity for samples assayed with varying concentrations ofcranberry juice. Ocean Spray 100% Cranberry and Concord Grape juiceblend containing 27% cranberry juice was used. In the absence of juice,background intensity was 67% of the total signal. Addition of 50% juiceblend (equivalent to 13.5% cranberry juice) reduced the backgroundintensity to less than 1% of the total signal (Johnson-White). Spikingsamples with grape juice was not found to produce this effect on thebackground signal (Welch's Purple 100% Grape Juice). Spiking of sampleswith apple juice, orange juice, and even white cranberry juice (OceanSpray 100% Juice Blend) also did not result in reduction of backgroundsignals. White cranberry juice is produced from cranberries harvestedearly before the red color and tart flavor are developed. The whitecranberry juice blend used contains 13.5% cranberryjuice. Thisdifference in concentration was accounted for when samples were spikedso that concentrations similar to those used with the red cranberryjuice were investigated.

Several mechanisms have been described for the inhibition of bacterialcell adhesion by cranberry juice (Steinberg et al., J. Antimicrob.Chemoth., 54, 86 (2004); Brumfelt et al., Lancet, 1, 186 (1962); Klepseret al., J. Infect. Dis. Pharmaco., 6, 1 (2003)). The acidity of thejuice, the sugar content including the rare D-mannose component, and thepresence of a rare polyphenolic component have been proposed ascontributing factors. Controlling the pH of the juice spiked sampleseliminated acidity as a causative factor. The sugar content of the juicewas eliminated as a factor through spiking of E. coli samples withsimilar concentrations of fructose, glucose, and mannose. Waveguidesurface passivation was also eliminated as a potential mechanism throughstudies of the impact of sample spiking on advancing contact angle.Though the contact angle was strongly impacted on clean glass slideswhen standard bacterial cell preparations were spiked, there was nonoticeable impact on the protein coated slides used in the ArrayBiosensor. The interaction of E. coli with human epithelial cells in theurinary tract is inhibited by A-type proanthocyanidins from cranberryjuice through interference with the p-fimbriae proteins on the bacterialcell surface (Liu et al., Biotechnol. Bioeng., 93, 297 (2006); Howell etal., Faseb J., 15, A284 (2001)). In order to investigate the potentialimpact of PACs on adhesion to the glass waveguides, sugars and othersmall molecules were eliminated from cranberry juice (Langer's CranberryConcentrate) through dialysis against water (Spectra/Por Membrane MWCO6-8000) ((Steinberg et al., J. Antimicrob. Chemoth., 54, 86 (2004).Colloidal particles were removed through filtration of the dialyzedmaterial using a 0.2 μm filter (Acrodisc PF; Gelman Sciences, Ann Arbor,Mich.). The dialyzed material was reconcentrated under nitrogen to a 27%cranberry juice equivalent and used to spike samples for Array Biosensorassays. This dialyzed filtered cranberry juice produced results similarto those observed when samples were spiked with the cranberry juiceblend.

EXAMPLE 3

LPS binding assays—The ability of both of PACs to inhibit theinteraction of LPS with PB was assessed using an agarose bead-basedpull-down assay (FIGS. 3, 4). Polymyxin B (10 μM, conjugated to agarosebeads (Sigma)) was incubated with 100 nM LPS-FITC (E. coli serotypeB5:055, Sigma) in the absence or presence of DCC, non-dialysed LH20 PAC,or size-fractionated LH20 PAC in a final volume of 250 μL 0.05 M Trisbuffer (pH 8.5). Reactions were stirred for 1 h at 25° C. in the dark.Unbound LPS-FITC was removed by three rounds of centrifugation andwashing with 250 μL of 0.05 M Tris buffer, followed by resuspension in200 μL of nuclease free water. Serial dilutions of each sample wereprepared in nuclease free water and the fluorescence was measured byexcitation at 495±2.5 nm and emission at 535+2.5 nm using a Saphirefluorescence plate reader (Tecan, Durham, N.C.). Comparable experimentswere performed with LPS from Salmonella, Shigella, and Pseudomonas andLPS from mutant strains of Salmonella minnesota (Rc mutant) and E. coliEH 100 (Ra mutant). The latter two strains contain polysaccharide chainsof varying lengths relative to native LPS. Binding experiments were alsoperformed with diphosphoryl lipid A. Conjugation of lipid A and LPS wasperformed with fluorescein isothiocyanate (Sigma) per the manufacturer'sinstructions and materials were subjected to dialysis against PBS forseparation from unbound dye. In all cases, the degree of conjugation wasdetermined by spectroscopy to be approximately 2-3 fluoresceins per moleof labeled species.

Using a solid-phase binding assay, the ability of both A- and B-typePACs to bind LPS was assessed by determining their ability to inhibitthe interaction of E. coli LPS with immobilized polymyxin B. Bindingexperiments comparing PACs from cranberries (which possess both A- andB-type linkages) to those from tea and grapes (which possess exclusivelyB-type linkages) demonstrated that PACs bearing both linkages boundefficiently to LPS in a dose-dependent manner (FIG. 4(A)). When thesedata were plotted as percent inhibition, cranberry PACs that had beenenriched through dialysis to contain polymers of larger molecular weightexhibited the most potent LPS binding activity with an IC₅₀ of 0.7 μM(FIG. 4(B)). PACs from tea (non-dialyzed) were the next most active withan IC₅₀ of 1.1 μM. Non-dialyzed PACs from grapes and cranberriesexhibited comparable relative affinities for LPS (IC₅₀=3.0 μM). Dialyzedcranberry concentrate (not enriched for PACs) exhibited the lowestrelative affinity for LPS (IC₅₀=10.5 μM). When the LPS bindingactivities of cranberry PACs produced by differential dialysis werecompared, a positive correlation between the relative affinity for LPSand PAC molecular weight was observed. Indeed, the larger molecularweight polymers exhibited higher LPS binding activity relative to thelower molecular weight PACs (FIG. 4(C)). Thus, all subsequentexperiments were performed using the PAC fraction from cranberries withan average degree of polymerization of twenty-one (heretofore referredto as “cranberry PACs”).

The LPS binding activity of cranberry PACs was not limited to E. coliLPS as evidenced by their ability to bind with comparable affinities toLPS from Salmonella, Shigella, and Pseudomonas. Further, cranberry PACsbound to two LPS mutants bearing shorter polysaccharide chains ofvarying lengths (an Ra mutant from E. coli and an Rc mutant fromSalmonella) with only a three-fold lower affinity relative to wild-typeLPS. These results are summarized in Table 2.

TABLE 2 Binding of cranberry LH20 PAC^(a) to LPS and Lipid A ApparentIC₅₀ Apparent IC₅₀ Bacterial species (μM)^(b) - LPS (μM)^(b) - LipidA^(c) Escherichia coli 0.7 ± 0.2 0.3 ± 0.1 Salmonella minn. 1.2 ± 0.3Shigella flexneri 1.6 ± 0.3 Escherichia coli EH 100 (Ra mutant) 2.1 ±0.7 Salmonella minn. (Rc mutant) 2.1 ± 0.5 Pseudomonas aeruginosa 3.4 ±1.1 ^(a)Corresponds to PACs of greater than 6,000 molecular weight.^(b)Apparent IC₅₀s are shown with their corresponding 90% confidenceintervals. ^(c)Diphosphoryl form of lipid A

As polymyxin B is known to bind to the lipid A portion of LPS (David etal., Biochem. Biophys. Acta., 1165, 147-152 (1992)), it was reasonedthat interaction with the lipid A moiety plays a predominant role incranberry PACs' recognition of LPS. Indeed, cranberry PACs efficientlyinhibited the binding of E. coli lipid A to polymyxin B with an apparentIC₅₀ of 0.3 μM, a relative affinity that is only two-fold greater thanits affinity for intact E. coli LPS (Table 2). This result confirms theimportance of the lipid A moiety in cranberry PACs' binding to LPS.

EXAMPLE 4

Analysis of LPS membrane binding and endocytosis. Human embryonic kidneycells (HEK 293) stably expressing human CD14 and TLR4/MD2(HEK-CD14-TLR4/MD2; Invivogen) were grown in chambered wells andincubated with 25 nM LPS (E. coli serotype 055:B5, Sigma) in the absenceor presence of 0.5 μM LH20 PAC (Dial>6K) for 1.5 h at 37° C. In controlexperiments, TLR4 or CD14 was functionally blocked by co-incubation withan anti-TLR4 or anti-CD14monoclonal antibody (500 nM in binding sites,Abeam, Inc.) or lipid A (Sigma). After incubation, the cells were washedwith PBS (10 min) twice and either fixed (with 3.7% paraformaldehyde) toassess LPS membrane binding or fixed and permeabilized (with 0.1% TritonX-100) to determine LPS internalization. After blocking with 1% normalgoat serum, membrane bound or internalized LPS was detected using a goatanti-LPS antibody (O/K serotype-specific, Abeam) conjugated tofluorescein. Nuclei were counterstained with DAPI. Imaging was performedusing an Olympus IX-71 microscope. The relative amounts ofmembrane-associated or intracellular fluorescence were quantified byimage analysis using Image J software (NIH, v. 1.37). Data is reportedas the mean channel fluorescence from membrane-associated orinternalized LPS and represents the analysis of 10 to 20 cells from eachsample (minimum 10 measurements per each cell). Merged images wereproduced using Photoshop CS2 (ver. 9).

PACs Slightly Inhibit Membrane Binding of LPS and Significantly InhibitLPS Endocytosis. Beyond the mere binding of LPS, a desirable attributeof LPS-binding compounds is the ability to inhibit LPS interaction withLPS-responsive mammalian cells. Based on their potent LPS-bindingactivity, it was reasoned that cranberry PACs could potentially inhibitLPS interaction with cells expressing the full complement of LPSreceptors. Cellular binding studies performed in HEK 293 cellsexpressing CD14 and Toll-like receptor 4/MD2 (HEK-CD14-TLR4/MD2),revealed a distinct staining pattern corresponding to membrane-bound LPS(FIG. 5(A), frame “LPS”) with minimal nonspecific binding (FIG. 5(A),frame “No LPS”). While co-incubation of LPS with lipid A did notsignificantly reduce LPS membrane binding, the presence of cranberry PACresulted in a modest but significant decrease (˜15%) in the amount ofmembrane-bound LPS (FIG. 5(C)). An anti-TLR4 function-perturbingantibody also caused a modest decrease in LPS binding (˜23%), while thissame antibody in combination with cranberry PAC did not impart anyfurther LPS binding perturbation. It was found that an anti-CD14function-perturbing antibody mediated the largest degree of LPS bindinginhibition (˜84% inhibition), demonstrating the highly important role ofCD14 in LPS membrane binding. Analysis of LPS internalizationdemonstrated that lipid A and cranberry PAC significantly inhibitedendocytosis of LPS (FIG. 5(B)) with degrees of inhibition of 84% and76%, respectively (FIG. 5(D)). The anti-TLR4 antibody mediated ˜50%inhibition of LPS endocytosis while co-incubation of the antibody withcranberry PAC increased this inhibition further to ˜62%. The anti-CD14antibody mediated approximately 80% inhibition of LPS endocytosis. Incontrol experiments, the addition of Alexa Fluor 647-labeledtransferrin, a marker of the endocytotic pathway, to the culture mediumcontaining PACs and LPS showed that PACs had no inhibitory effect onnormal endocytosis as a robust staining of the endosomal compartment wasobserved in both the presence and absence of PACs. Thus, PACsspecifically inhibited the endocytosis of LPS while having no inhibitoryeffect on overall endocytosis (FIG. 6).

In the current model of cellular interaction with LPS, LPS-bindingprotein (LBP) present in serum binds to and presents LPS to themembrane-resident receptor CD14, which in turn transfers LPS to thebipartite receptor complex, TLR4/MD2 (Shimazu et al., J. Exp. Med.,1999, 189, 1777-1782). MD2 is the LPS-binding unit of the receptor whileTLR4 serves as the signal transduction component (Shimazu; Nagai et al.,Nat. Immunol., 2002, 3, 667-672; Schromm et al., J. Exp. Med., 2001,194, 79-88). The TLR4/MD2-LPS complex ultimately undergoes endocytosisinvolving a caveolae-dependent uptake mechanism as part of LPS-inducedreceptor down-regulation (Shuto et al., Biochem. Bioph. Res. Co., 2005,338, 1402-1409). While debate currently exists as to whether TLR4physically contacts LPS, it is clear that TLR4/MD2 and LPS form a stablecomplex on the cell surface and that LPS binding to MD2 is aprerequisite for TLR4 signaling activity (Visintin et al., J. Immunol.,2005, 175, 6465-6472) and LPS endocytosis (Shuto; Husebye et al., EMBOJ., 2006, 25, 683-692). It was hypothesized, therefore, that cranberryPACs inhibit LPS endocytosis by inhibiting LPS interaction with theTLR4/MD2 complex.

PAC Inhibition of LPS binding to LBP, CD14 and TLR4/MD2. Human CD14(Cell Sciences) was adsorbed onto ELISA plates in PBS overnight at 4° C.Histidine-tagged-human TLR4/MD2 (R&D Systems) or human LPS-bindingprotein (LBP, Biometec) was captured overnight at 4° C. onto ELISAplates prepared by the passive adsorption of anti-polyhistidinemonoclonal antibody (R&D Systems). Wells were blocked for 30 min at 37°C. with 1% normal goat serum in PBS. Binding of 5 nM E. coli LPS-FITCwas performed for 30 min at 37° C. in 1% fetal bovine serum in PBS inthe presence or absence of LH20 PAC. Soluble CD14, when present, was ata final concentration of 25 nM. Bound LPS-FITC was detected using a goatanti-fluorescein-horseradish peroxidase conjugate (Abcam) andtetra-methylbenzidine substrate (Kierkegaard and Perry). In the absenceof serum, binding of LPS-FITC to CD14 or to TLR4/MD2 was below thedetection limit.

PACs Abrogate LPS Interaction with CD14 and TLR4/MD2 but not LPS-BindingProtein (LEBP). Binding studies were performed in order to address theeffect of cranberry PACs on LPS interaction with its cognate receptors.FIG. 7(A) shows the results of binding experiments conducted to measurethe ability of cranberry PACs to inhibit the binding of E. coli LPS toimmobilized LBP, CD14, or TLR4/MD2. It was apparent that cranberry PACshad no significant effect on LPS interaction with LBP while theyachieved a maximum inhibition of 38% of LPS binding to CD14 at thehighest PAC concentration tested (500 nM). Over the same concentrationrange, cranberry PACs completely inhibited LPS interaction withTLR4/MD2, with an IC₅₀ of 20 nM PAC. It was further found that in thepresence of soluble CD14, the amount of LPS bound by TLR4/MD2 wasincreased approximately four-fold (FIG. 7(B)), consistent with theestablished role of CD14 in mediating the transfer of LPS to TLR4/MD2(Aderem et al., Nature, 2000, 406, 782-787; Medzhitov, Nat. Rev.immunol., 2001, 1, 135-145). The degree to which cranberry PAC inhibitedLPS binding to TLR4/MD2, however, remained unchanged demonstrating theability of PACs to inhibit the CD14-mediated transfer of LPS toimmobilized TLR4/MD2 (FIG. 7(B), inset).

Quantification of NF-κB activation. HEK-CD14-TLR4/MD2 cells weretransiently transfected with the NF-κB-inducible reporter plasmid,pNiFty2-SEAP (Invivogen), which encodes secreted embryonic alkalinephosphatase (SEAP) under the control of a 5×NF-κB-inducible promoter.Cells were seeded into wells of a 96-well plate (4×10⁴ cells/well) andtransfected using Effectene reagent (Qiagen) per manufacturer'sinstructions. After 48 h, the cells were stimulated for 16 h with 2 nMLPS in the presence or absence of cranberry PACs. SEAP activity wasmeasured in tissue culture supernatants using a colorimetric SEAP assaykit (Invivogen) according to the manufacturer's protocol.

PACs Inhibit LPS-induced NF-κB Activation. Based on the LPS-bindingactivity of cranberry PACs and their abrogation of LPS interaction withcell surface receptors, it was reasoned that PACs could also inhibit theLPS-induced activation of the transcription factor, NF-κB. NF-κBactivation by LPS leads to the expression of proinflammatory cytokines,resulting in the metabolic and physiologic changes that ultimately leadto pathological conditions, including sepsis (Baeuerle et al., Ann. Rev.Immunol., 1994, 12, 141-179). As shown in FIG. 8(A), cranberry PACsinhibited the activation of NF-κB in a dose-dependent manner inHEK-CD14-TLR4/MD2 cells stimulated with 2 nM LPS, with an IC₅₀ of 25 nMPAC. Further, the data in FIG. 8(B) show that this inhibition was notreadily overcome by an excess of LPS. In the absence of cranberry PAC,an increase in LPS resulted in a corresponding increase in the NF-κBresponse. When the LPS concentration was increased above 2 nM, a slightdecrease in the response was noted, due primarily to LPS-inducedcytotoxicity (see below). In the presence of 0.5 μM cranberry PAC, aconsistent decrease in the NF-κB response was observed across all LPSconcentrations. Even when LPS was present at 3 nM (a six-fold molarexcess over PAC), NF-κB activation was not restored to control levels.When cranberry PAC was present at 10 nM, a consistent decrease in theNF-κB response (approximately 50% across all LPS concentrations) wasobserved relative to the control.

EXAMPLE 5

Cytotoxicity assays—Cellular toxicity was measured using a calorimetriccell proliferation assay (CellTiter96™, Promega). HEK-CD14-TLR4/MD2cells were seeded into the wells of a 96-well plate (1×10⁴ cells/well)and cultured with a dose range of test compounds for 48 h prior to assayaccording to the manufacturer's instructions. Examination of PACs'cytotoxicity revealed an IC₅₀ for toxicity of 700 nM, with no toxiceffects observed at concentrations below 100 nM (FIG. 8(C)). Whencompared to LPS (IC₅₀ for toxicity of ˜6 nM), PACs were more than100-fold less toxic. In comparison to native LPS, the diphosphoryl formof lipid A did not elicit toxicity at concentrations below 3 μM.

EXAMPLE 6

Immobilization of PACs and LPS binding assay—To determine the ability ofPAC materials to effectively bind bacteria and bacterial cell componentsupon immobilization to a solid support, PACs from cranberry and grapejuices were covalently attached to the surface of a glass microscopeslide (waveguide) using a modification of a common technique used forprotein immobilization (Rowe et al., Anal. Chem., 71, 3846 (1999)).Catechin monomer and buffer only areas were also included as controls.Briefly, the waveguides, glass microscope slides (Daigger, Wheeling,Ill.), were cleaned by immersion in potassium hydroxide/methanolsolution (Cras et al., Biosens. Bioelectron., 14, 683 (1999)) followedby rinsing and drying. The clean waveguides were then incubated with 2%(3-mercaptopropyl)triethoxysilane (Pierce Chemicals, Rockford, Ill.) intoluene for 45 minutes followed by rinsing and drying. The slides wereimmersed in 1.8 mM N-[p-maleimidophenyl]isocyanate (PMPI) in ethanol for1 hour. The maleimide-group of PMPI reacts with the sulfhydryl-groupprovided by the silane reaction leaving the isocyanate-group free toreact with the hydroxyl-groups of PACs. Following PMPI immobilization,waveguides were rinsed with deionized water, dried, and mounted in PDMSpatterning templates described previously (Rowe). Solutions of 10 mg/mLPACs from cranberry or grape juices, catechin in 10 mM PBS with 10%methanol, and PBS with 10% methanol (negative control) were incubated inthe lanes of the PDMS template overnight at 4° C. Patterning solutionswere flushed from the PDMS lanes using PBS and the slides were rinsedwith deionized water, dried, and stored at 4° C. until use. The abilityof the immobilized PACs to bind LPS was investigated using a stationaryassay. PDMS flow cells with channels were affixed perpendicularly tothose of the patterning template so that each of the sample lanes wasexposed to each of the patterned rows. Sample lanes were rinsed with PBSfollowed by injection of various concentrations of LPS-FITC (E. coli055:B5, Sigma) in PBS. The LPS-FITC was incubated in the lanes for 1hour, followed by rinsing with PBS and deionized water. Slides wereimaged using evanescent wave fluorescence spectroscopy with excitationat 496 nm and emission collected above 515 nm. Upon exposure to aconcentration range of LPS-FITC, it was apparent that PACs fromcranberry and grape juices bound specifically to LPS-FITC while thecatechin and buffer controls show no binding even at the highestconcentrations tested. Thus, PACs retain their ability to bind LPS uponimmobilization to a solid support.

EXAMPLE 7

PAC and catechin immobilization—Purified PACs and catechin(Sigma-Aldrich) were immobilized onto activated thiol-Sepharose® 4B(Sigma-Aldrich) via a (N-[p-maleimidophenyl]isocyanate) (PMPI; Pierce)crosslinker. Sepharose® was swelled in deionized water for 1 hour (1 gdry material in 15 mL H₂O). The material was then washed using freshdI-H₂O over five suspend/centrifuge/decant cycles (total 50 mL per gramdry starting material). Suspension was accomplished using a vortex andcentrifuge steps were conducted at 3,000 g for five minutes. TheSepharose® material (total volume 5 mL per gram starting material) wasthen rinsed three times with ethanol (total volume 30 mL per gramstarting material). The PMPI crosslinker was incubated with the rinsedSepharose material for 1 hour at room temperature under constantagitation using a 10-fold molar excess of PMPI over the thiol-groupconcentration with 3% dimethylsulfoxide in ethanol (10 mL per gramstarting material). Incubation was followed by centrifuging, decanting,and rinsing in 50% ethanol for three cycles. The Sepharose was thenincubated overnight at 4° C. in ethanol with PAC or catechin using a10-fold molar excess of analyte in 50% ethanol (10 mL per gram startingmaterial). As a final step, the Sepharose was rinsed over four cyclesusing 50% ethanol (40 mL total per gram starting material) andresuspended in 0.02% sodium azide in H₂O (16 mL final volume per gramstarting material). The materials were stored in the dark at 4° C. untiluse. PAC concentrations for bead sets were determined by Prussian blueassay.

EXAMPLE 8

Fluorescence-based pull down assays for lipopolysaccharide (LPS) wereconducted in 50 mM TRIS at pH 8.0 using Sepharose-immobilized PACs andagarose-immobilized polymyxin B (Sigma-Aldrich, St. Louis, Mo.).Immobilized capture molecules and LPS were incubated at room temperaturefor 1 hour with constant agitation followed by rinsing and transfer to a96-well plate. The fluorescence of the FITC-labeled LPS (from E. coli055:B5; Sigma-Aldrich) was measured using a Tecan XSafiremonochrometer-based micro plate reader at 495 nm excitation and 520 nmemission (5 nm bandwidths).

PAC beads for use in pull-down assays were generated by immobilizingproanthocyanidins from cranberries, cranberry juice, tea, and grapejuice onto Sepharose beads. An additional bead set was generated byimmobilizing the fraction of PACs from cranberries with molecularweights greater than 6,000 onto Sepharose beads. Assays conducted usingthese beads demonstrated that all five bead sets could be used to bindFITC-labeled LPS from solution (50 mM TRIS pH 8). Concentrationdependence curves for beads with immobilized PACs from tea andcranberries are presented in FIG. 9. Beads coated with catechin wereused to verify a low degree of nonspecific binding of FITC-LPS to theSepharose and crosslinker. In order to further verify that binding ofLPS to the Sepharose beads was via a specific interaction with theimmobilized PAC, a competitive assay was used in which soluble PAC wasadded to the sample solution during the assay. Increasing theconcentration of soluble PAC was found to decrease the fluorescenceintensity resulting from the pull-down assay as expected (FIG. 10).

In FIG. 10 soluble PACs from tea have been added to assays conductedwith immobilized PACs from tea while soluble PACs from cranberries havebeen added to assays conducted with immobilized PACs from cranberries.Inhibition of 50% of LPS binding by the PAC-tea beads occurs at 6.5 μMwhile 50% inhibition of binding by PACs from cranberries occurs at 9.8μM. Capture molecule concentration in the two assay types is 5.5 μM forPAC-tea beads and 6.0 μM for PAC-cranberry beads. The discrepanciesbetween the capture molecule concentration and the level at which 50%inhibition occurs are likely due to several considerations. Capturemolecule concentrations have been estimated using the Prussian blueassay as compared to the Prussian blue assay conducted on soluble PACsfrom the same source. This assignment assumes that all components of theimmobilized polyphenolics are accessible in a maimer similar to those insolution. The analysis also assumes that all degrees of polymerizationwere immobilized equally so that the degree of polymerization achievedon the Sepharose was similar to that observed in solution. Thoughrelatively low soluble PAC concentrations were not found to quench thefluorescence of FITC-LPS, immobilized PACs represent a locally highconcentration. The impact can be seen in FIG. 9. The increase influorescence intensity with increasing target concentration cannot bedescribed by a simple model. In addition, although PAC absorbance in thewavelength range of interest is low with molar extinction coefficientson the order of 10,000, the locally high concentration of PACs reducesthe excitation intensity available to the FITC-LPS. All of these factorsmay contribute to either an actual difference, as with the Prussian blueassay, or to an observed difference, as with the quenching, in theexpected concentration of soluble PACs needed to obtain 50% inhibitionof LPS binding by the immobilized PACs.

Though inhibition of the PMB-LPS interaction (Example 3) indicated theinteraction of PACs with LPS in a manner which prevented PMB binding, itdid not guarantee the interaction of PACs with the lipid A portion ofLPS. To further investigate this potential interaction, an assay for thepresence of FITC-LPS using PAC-beads was conducted after equilibrationof the PAC beads with varying concentrations of lipid A (FIG. 11).Assays were conducted with 3 μg/mL FITC-LPS (approx. 3 nM). The presenceof lipid A was found to inhibit FITC-LPS binding by immobilized PACswith IC₅₀ values of 100 nM and 500 nM for PACs from tea and cranberries,respectively. The addition of lipid A concentrations as high as 100 μM,however, failed to result in inhibition of greater than 80% of theFITC-LPS binding. It is unclear whether this is a result of theunavailability of lipid A, which may form micelles or precipitate at thehigher concentrations, or of the fact that binding interactions betweenLPS and the immobilized PACs are not limited to the lipid A portion ofLPS.

In order to evaluate the utility of Sepharose-immobilizedproanthocyanidins for LPS capture, a side-by-side comparison was made tothat of commercially available agarose-immobilized polymyxin B. FIG. 12presents the percent LPS captured as a function of the concentration ofcapture molecule for PMB and PACs from tea and cranberries. Quenching ofthe FITC-LPS fluorescence intensity when bound to the immobilized PACpresented a difficulty in direct comparison of the capture materials.This issue was addressed by measuring the fluorescence intensity of theFITC-LPS remaining in solution after incubation with the capturematerial. Percent captured values are based on comparison of thefluorescence remaining after incubation with capture material to anidentically handled sample which contained no capture molecule. Bothtypes of PAC beads performed slightly better than the PMB beads. Thiswas expected based on the inhibition of polymyxin B binding of LPS(Example 3) by the proanthocyanidins.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that the claimed invention may be practiced otherwise than asspecifically described. Any reference to claim elements in the singular,e.g., using the articles “a,” “an,” “the,” or “said” is not construed aslimiting the element to the singular.

1. A composition comprising: a proanthocyanidin; and a macromolecule, anassembly of macromolecules, a semi-solid, or a solid surface to whichthe proanthocyanidin is immobilized.
 2. The composition of claim 1,wherein the macromolecule comprises an amino acid, a peptide, a protein,a nucleotide, a nucleic acid, a lipid, or a carbohydrate.
 3. Thecomposition of claim 1, wherein the assembly of macromolecules comprisesa multi-protein complex, a virus, a dendrimer, or a nanoparticle.
 4. Thecomposition of claim 3, wherein the nanoparticle comprises ananocluster, a nanocrystal, a nanorod, a nanosphere, or a nanotube. 5.The composition of claim 1, wherein the semi-solid or solid surfacecomprises a plurality of beads, a sphere, a rod, fibers, filaments,capillaries, a tube, a planar layer, or a waveguide.
 6. A filtercomprising: a housing having an inlet and an outlet to allow fluid flowthrough the housing; and the composition of claim 1; wherein thecomposition is maintained within the housing when fluid is flowedthrough the housing.
 7. A sensor comprising: the composition of claim 1;a fluid flow apparatus; and a mechanism for detecting the binding oflipopolysaccharide, lipid A, or bacteria to the proanthocyanidin.
 8. Thesensor of claim 7, wherein detection is achieved through an opticalmechanism, ultraviolet light absorbance, visible light absorbance,infrared absorbance, fluorescence, luminescence, chemiluminescence,polarization, surface plasmon resonance, or changes in refractive index.9. The sensor of claim 7, wherein detection is achieved through anacoustic mechanism, a surface acoustic wave device, or a quartz crystalmicrobalance device.
 10. The sensor of claim 7, wherein detection isachieved through an electrochemical mechanism, or amperometric,potentiometric, or conductimetric measurements.
 11. The composition ofclaim 1, wherein the average degree of polymerization of all theproanthocyanidin in the composition is at least about
 6. 12. Thecomposition of claim 1, wherein the average degree of polymerization ofall the proanthocyanidin in the composition is from about 6 to about 40.13. The composition of claim 1, wherein the average degree ofpolymerization of all the proanthocyanidin in the composition is fromabout 20 to about
 22. 14. The composition of claim 1, whereinproanthocyanidin consists of catechin and epicatechin units.
 15. Amethod comprising: providing the composition of claim 1; and exposingthe composition to a sample suspected of containing alipopolysaccharide, lipid A, or a bacterium that produceslipopolysaccharide or lipid A.
 16. The method of claim 15, wherein thecomposition is located within a housing having an inlet and an outletfor fluid flow through the housing.
 17. The method of claim 15, whereinthe composition is located within a fluid flow apparatus that allows forthe detection of binding of lipopolysaccharide, lipid A, or bacteria tothe proanthocyanidin.
 18. The method of claim 15, further comprising:detecting the binding of the proanthocyanidin to the lipopolysaccharide,lipid A, or bacterium through an optical mechanism, NV absorbance,visible absorbance, infrared absorbance, fluorescence, luminescence,polarization, surface plasmon resonance, or changes in refractive index.19. The method of claim 15, further comprising: detecting the binding ofthe proanthocyanidin to the lipopolysaccharide, lipid A, or bacteriumthrough an acoustic mechanism, a surface acoustic wave device, or aquartz crystal microbalance device.
 20. The method of claim 15, furthercomprising: detecting the binding of the proanthocyanidin to thelipopolysaccharide, lipid A, or bacterium through an electrochemicalmechanism, amperometric, potentiometric, or conductimetric detector. 21.The method of claim 15, wherein the average degree of polymerization ofall the proanthocyanidin is at least about
 6. 22. The method of claim15, wherein the average degree of polymerization of all theproanthocyanidin is from about 6 to about
 40. 23. The method of claim15, wherein the average degree of polymerization of all theproanthocyanidin is from about 20 to about
 22. 24. The method of claim15, wherein the sample is a clinical sample.
 25. The method of claim 24wherein the clinical sample comprises blood, plasma, serum, lymph, orspinal fluid.
 26. The method of claim 15, wherein the sample is apharmaceutical preparation.
 27. The method of claim 15, wherein thesample is a food or beverage intended for infants or immunosuppressedindividuals.